A successful biological transformation of an organism must satisfy the following three criteria:
1. Transforming DNA must enter the organism by physical or chemical means such as electrotransformation, treatment with inorganic ions, protoplast fusion, etc. PA1 2. Transformants must be selected with the help of one or more markers from the non transformed cells in the population for instance by antibiotic resistance genes linked to the transforming DNA. This is best satisfied by either the isolation of a resistance gene against an antibiotic from the target host in question, or by the engineering of a known resistance gene with expression sequences (promoter and terminator) compatible with the target host. PA1 3. Transforming DNA must be replicated (either autonomously or as part of the host genome). This is best satisfied by the isolation of replicating plasmids from the host to be transformed and to subsequently construct vectors able to replicate in a microorganism such as Escherichia coli (E. coli) or Lactococcus lactis (Lc. lactis) and in a specific target organism such as Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). PA1 resistant strains to phages, PA1 ropy strains (improved texturing properties), PA1 probiotic strains, PA1 strains producing new or improved enzymes (lipases, deshydrogenases, . . . ), aroma or flavor compounds, . . .
The international patent application WO92/14825 describes a plasmid pBULI having a length of about 7.9 kb and its derivative isolated from Lactobacillus delbrueckii subsp. bulgaricus M-878 strain.
The restriction map of this plasmid is characterized by the absence of restriction sites for BamHI, EcoRI, KpnI and PstI enzymes.
This plasmid is used as a vector for breeding various microorganisms such as lactic acid bacteria and the derivative of this plasmid is used as a shuttle vector (lactic acid bacterium--Escherichia coli).
Other shuttle vectors are described in the documents Canadian Journal of Microbiology (vol. 38 (1992) pp 69-74), ACTA MICROBIOLOGICA BULGARICA (vol. 27 (1991) 99 3-8) and in the Japanese Patent Application JP-A-4.218.381.